FGF-2通过β-连环蛋白-LEF-1调控CTEPH肺动脉内皮间充质转化中的作用及机制

Chronic thromboembolic pulmonary hypertension (CTEPH) has high morbidity and mortality. Pulmonary artery endothelial cell (PAEC) mesenchymal transition (EndMT) has been found to be involved in endothelial remodeling in clinical CTEPH endarterectomy specimen. Because of the lack of reliable CTEPH animal model, the exact and detail mechanism of EndMT remains unknown.It is known that fibroblast growth factor (FGF-2) and Wnt signaling pathways are involved in the EndMT. During the EndMT of the endocardium, the canonical Wnt/ beta catenin (Wnt/β-catenin) is a key signaling pathway, and the lymph enhancement factor -1 (LEF-1) is the key point of downstream of EndMT followed with the β-catenin. We have established a reliable CTEPH rat model, and found that PAEC had involved with obvious EndMT in the model, also with the expression of FGF-2 and LEF-1 increasing significantly. Therefore, we assume that FGF-2 may regulate β-catenin by classical Wnt/β-catenin signal pathway or bypass signal pathway, thus affecting the LEF-1 activity, leading to the EndMT transformation of PAEC in CTEPH . Because the EndMT may be reversed, we have ideas to study further.We will collect the CTEPH clinical specimens and combined with the animal experiments to detect the alpha -SMA and CD31 confirming the EndMT of PAEC with the analysis of PCR array technology. We will detect the changing of the β-catenin with the application of different concentrations of FGF-2 and observe the structural and functional changes of PAEC and EndMT through the highly selective inhibiting the key points of Wnt, β-catenin, LEF-1 transfected with siRNA,knocking out LEF-1 gene with Crispr/cas9 genome editing methods from many aspects and levels such as cell, tissue, animal levels to interfere the signal pathway, to provide new ideas and methods for the prevention and early intervention of CTEPH development.

慢性血栓栓塞性肺动脉高压（CTEPH）病死率高。内膜剥脱术后标本发现肺动脉内皮间充质转化（EndMT），但因缺乏深入研究动物模型，机制不明。已知淋巴增强因子（LEF）-1是Wnt/β-连环蛋白（β-catenin）通路调节EndMT的下游关键点。我们建立可靠的CTEPH大鼠模型发现肺动脉内皮成纤维生长因子(FGF)-2和LEF-1表达升高且发生了EndMT。因此假设，FGF-2通过经典Wnt/β-catenin通路或旁路影响β-catenin-LEF-1而调控CTEPH的EndMT。我们拟用PCR阵列技术检测α-SMA与CD31分析EndMT；予FGF-2不同浓度干预；从细胞、组织、动物水平对上述通路关键点如Wnt、β-catenin、LEF-1等高选择性抑制或激动、siRNA干扰抑制及Crispr/cas9 敲除LEF-1基因等，观察内皮EndMT和功能变化，为防治CTEPH提供新思路。

慢性血栓栓塞性肺动脉高压（CTEPH）病死率高。内膜剥脱术后标本发现肺动脉内皮间充质转化（EndMT），但因缺乏深入研究动物模型，机制不明。已知淋巴增强因子-1（LEF-1）是Wnt/β-连环蛋白（β-catenin）通路调节EndMT的下游关键点。我们建立可靠的CTEPH大鼠模型发现肺动脉内皮成纤维生长因子-2（FGF-2）和LEF-1表达升高且发生了EndMT。.本研究主要研究CTEPH动物模型的建立、肺栓塞慢性化至CTEPH肺动脉EndMT，假设FGF-2通过经典Wnt/β-catenin通路或旁路影响β-catenin-LEF-1而调控CTEPH的EndMT。予FGF-2不同浓度干预；从组织、动物水平对上述通路关键点β-catenin高选择性抑制或激动，观察内皮EndMT和功能变化，为防治CTEPH提供新思路。.在上述实验中，通过我们成功建立的CTEPH大鼠模型。CTEPH大鼠肺动脉压力随着栓塞时间的延长而增加，内皮明显增生，发生间充质转化即内皮细胞特征标志物CD31、闭合蛋白（Occludin）表达减少，间质细胞标志物α-SMA、波形蛋白（Vimentin）表达增加。发现FGF-2表达与mPAP、LEF-1、Vimentin呈正相关，与Occludin呈负相关；LEF-1表达与平均肺动脉压（mPAP）、Vimentin呈正相关，与Occludin呈负相关。提示FGF-2和LEF-1在CTEPH大鼠肺动脉EndMT中发挥重要作用。.我们进一步研究探究了β-catenin在CTEPH大鼠肺动脉EndMT中的作用。通过β-catenin特异性抑制剂干预CTEPH大鼠，发现随着栓塞时间的延长而增加，干预组较实验组相比：β-catenin、α-SMA、mPAP降低（P<0.05）；CD31表达升高（P<0.05），EndMT改善明显，表明抑制β-catenin的表达可能在CTEPH大鼠肺动脉EndMT中发挥重要作用。.最后，我们还研究FGF-2、Wnt/β-catenin通路对CTEPH肺动脉EndMT功能的影响。发现FGF-2抑制剂可以减弱Wnt/β-catenin表达，改善CTEPH大鼠肺动脉EndMT，降低CTEPH大鼠mPAP。FGF-2激动剂可以增强Wnt/β-catenin表达，加重CTEPH大鼠肺动脉EndMT，升高CTEPH大鼠mPAP。提示FGF-